Studies on Ultrastructural Identification and Distribution of Protein-Polysaccharide in Cartilage Matrix

Matukas, Victor J.; Panner, Bernard J.; Orbison, J. Lowell

doi:10.1083/jcb.32.2.365

Cited by 189 publications

(37 citation statements)

References 36 publications

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“…Sulfated GAG's are readily extracted from tissues by the usual fixation and dehydration methods; however, they can be retained in connective tissue when cationic dyes or reagents (such as cetyl pyridinium chloride [29], RR [28], alcian blue [1,47], or safranin O [44]) are added to the fixative. RR has been shown to bind to, to precipitate and to stain GAG's in a variety of connective tissues (15,16,34,48,50), where they occur as proteoglycan particles (20=50 nm in diameter) often closely associated with collagen fibrils (16,34,47,48,50).…”

Section: Nature Of the Anionic Sites In The Gbmmentioning

confidence: 99%

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Kanwar

Farquhar

1979

The Journal of Cell Biology

508

200

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Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (-60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (-3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells 9 When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites 9 CF remained firmly bound after prolonged perfusion with 0.1-0 9 M KCI or NaCI. It was displaced by perfusion with buffers of high ionic strength (0 9149 M KCI) or pH (<3.0 or >10.0). CF also bound (clustered at -60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix 9 The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations 9 KEY WORDS cationized ferritin red proteoglycans 9 ionic interaction isolated GBM's 9 ruthenium Recent studies have shown that the glomerular capillary wall contains fixed negatively charged sites which may be important in maintaining nor- J. CELL BIOLOGY 9The Rockefeller University Press 9

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Section: Nature Of the Anionic Sites In The Gbmmentioning

confidence: 99%

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Kanwar

Farquhar

1979

The Journal of Cell Biology

508

200

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“…These all showed darkly stained a p p a r e n t l y degenerating chondrocytes (Figure 5A), Cells were surrounded by lacunae containing stellate bodies and thin fibrils as in normal cartilage (13). There were some normal appearing matrix vesicles, but others were large, oval, o r irregular and very dense.…”

Section: Synovial Effusionmentioning

confidence: 98%

Ochronotic arthropathy with calcium pyrophosphate crystal deposition a light and electron microscopic study

Reginato

Schumacher

Martı́nez

1973

Arthritis & Rheumatism

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Synovial fluid from a patient with ochronotic arthropathy and calcification of the capsule of the right knee CGntained calcium pyrophosphate crystals and dark debris. Microscopic examination of an unstained fresh preparation showed these dark fragments t o have a brown or golden brown color typical of ochronotic pigment. By electron microscopy there were identifiable cartilage fragments in the synovial fluid. These contained clumps of dense material deposited on the surface of collagen fibers. Similar brown staining and electron dense material probably representing ochronotic pigment was seen in synovial membrane and synovial fluid cells. Some of the intracellular calcium pyrophosphate crystals were in close relation with probable pigment.Ochronosis is a well-known but uncommon cification of peripheral joints has been rare (1,2 705REGINATO E l AL light microscopic findings include degenerative mented inclusions in synovial fluid monochanges in cartilage, diffuse pigmentation of the nuclear cells (5). cartilage matrix, brown ochronotic pigment inThis report describes light and electron miside the chondrocytes and in phagocytes about croscopic findings in synovial membrane of a the joint detritus, and pigmented shards in sv-patient with ochronotic arthropathy and synovial membrane. Huttl has shown dark pig-novial or capsular calcification. We report for MATERIALS AND METHODSThe patient was a 65-year4d male with characteristic Oetrmnotic pimentation of sclerac and ears, and a n h r o p atthy involving knees, spine. and shoulders. A diapnosis of l k r p a n u r i a was established by the urinary excretion of 24 ofhomogentistic acid in 24 hours. T h e detailed clinical and ndidogic aspects of this case have bcen reported elsewhere (6). In 1969 the patient had typical radiolosic signs d ahronotic spondglosis. At that time knee roentqeno- bilateral. sliqhtly painful knee effusions without increased heat or erythema. Roentqenoqrams showed proqrnsion of the dcqenerative changes an0 appearance of r;llrification in the joint capsule and/or synovium (Fiqurc I ).Synovial fluid was aspirated from both knees and cxamincd by standard synovianalvsis (7). An aliquot was centrifuged and the sediment fixed in half-strenqth Karnovsky's fixative and processed as described below for electron mi- RESULTS Synovial EffusionThe synovial fluid was yellow and clear with many dark strands; mucin clot and viscosity were normal. There were 100 WBC's/cu mm (56% lymphocytes and 44% large mononuclear cells). An unstained, wet preparation of synovial fluid revealed black and brown pigmented pieces of cartilage and fibrillar and unidentifiabk material (Figure 2). These fragments stained deeper brown with Wrisht's stain and black with the Masson-Fontana stain. A few rod and rhomboid shaped weakly positive birefringent crystals were identified both intracellularly and extracellularly. There were insufficient crystals for x-ray diffraction study.

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“…A number of studies has established that the polygonal matrix granules are composed, at least in part, of precipitated proteoglycan aggregates. These studies include the following criteria: Alcian blue stains localize the proteoglycan in these granules (2,5,30,34); prior treatment of cartilage with testicular hyaluronidase or chondroitinase ABC reduces the size and number of these matrix granules (18,34); extraction of cartilage with 4 M guanidine hydrochloride removes more than half 294 THE JOURNAL OF CELL BIOLOGY" VOLUME 73, 1977…”

Section: Discussionmentioning

confidence: 99%

“…Of the constituents of the extracellular matrix, two macromolecules, cartilage-type collagen (19,36) and cartilage-specific proteoglycans (13,22), are synthesized by the differentiated chondrocytes. Ultrastructurally, this extracellular matrix consists of thin, 200-300 ,~ wide cartilage-type collagen fibrils and polygonal matrix granules of precipitated proteoglycan aggregates (1,6,18,25,(33)(34)(35).…”

mentioning

confidence: 99%

Defects in the cartilaginous growth plates of brachymorphic mice.

Orkin¹,

Williams²,

Cranley³

et al. 1977

The Journal of Cell Biology

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Homozygous brachymorphic (bm/bm) mice are characterized by disproportionately short stature. Newborn bm/bm epiphyseal cartilages are shorter than normal although the cells in the different zones of growth are relatively well organized. The extracellular matrix reacts poorly with stains specific for sulfated glycosaminoglycans. The ultrastructural appearance of the cartilage matrix indicates normal collagen fibrils; however, proteoglycan aggregate granules are smaller than normal and are present in reduced numbers, particularly in the columnar and hypertrophic zones of the growth plate. In addition, a prominent network of fine filaments, which are extractable in 4 M guanidine hydrochloride, are present in the bm/bm cartilage matrix. These findings suggest that a defect affecting the proteoglycan component of cartilage occurs in bm/bm mice.Numerous inherited disorders affect cartilage structure and function in man and other animals. Recently, we have studied cartilage from brachymorphic (bm/bm) mice. This mutant was first described by Lane and Dickie (12), and established by genetic studies as an autosomal recessive condition. Homozygous bm/brn mice are vigorous but have shortened long bones, shortened tails, and dome-shaped skulls. Such findings are consistent with a defect in cartilage development, and Lane and Dickie found the knee joints of 17-24 day old bin~bin mice to be shorter and thicker than normal, although chondrocytes in these epiphyseal growth plates retained good alignment (12).Cartilage is composed predominantly of one cell type, chondrocytes, surrounded by large quantities of extracellular matrix. Of the constituents of the extracellular matrix, two macromolecules, cartilage-type collagen (19, 36) and cartilage-specific proteoglycans (13,22), are synthesized by the differentiated chondrocytes. Ultrastructurally, this extracellular matrix consists of thin, 200-300 ,~ wide cartilage-type collagen fibrils and polygonal matrix granules of precipitated proteoglycan aggregates (1,6,18,25,(33)(34)(35).In examining the cartilaginous epiphyses from bm/bm mice, we have observed alterations in the morphological appearance of the epiphyseal growth plates at different stages of development, as well as differences in the extracellular matrix material which may be related to the abnormalities in tissue development found in this mutant.

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Studies on Ultrastructural Identification and Distribution of Protein-Polysaccharide in Cartilage Matrix

Cited by 189 publications

References 36 publications

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Ochronotic arthropathy with calcium pyrophosphate crystal deposition a light and electron microscopic study

Defects in the cartilaginous growth plates of brachymorphic mice.

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